The crosstalk between actin cytoskeleton and microtubule (MT) dynamics is of extreme biological importance, given the fact that many biological processes rely on the correct coordination between these two networks. We have obtained recent experimental evidence suggesting the existence of a novel link between MT dynamics and actin cytoskeleton remodelling, involving localised Rho GTPase signalling operated by complexes of the RhoGEF Trio and MT-associated proteins. The RhoGEF Trio is a master gene that plays important roles in cell migration, axon outgrowth and guidance. Until recently, Trio was best known for its role in actin cytoskeleton remodelling via the regulation of the Rho family of GTPases. Partner 1 has identified Trio-associated proteins that are linked to the control of MT dynamics, namely several members of the +TIP family, such as CLASP2 and EB1 and has demonstrated that Trio itself is a novel +TIP. Of note, Trio is the sole member of the mammalian RhoGEF family that displays the ability to bind MT plus-ends. Concomitantly, partner 2, who is working on the +TIP Navigator-1 (NAV1), has identified Trio as a NAV1 partner. These data linking Trio and proteins related to MT dynamics represent a breakthrough in the understanding of Trio functions. Drawing on their reciprocal data, partners 1 and 2 have initiated a collaboration to study the functional relationship between Trio and its MT-associated partners. Our data show that the MT-binding ability of Trio is critical for its function in neuronal differentiation, by specifying localised Rho GTPase signalling upon its recruitment to the plus-end of MTs. We have also gathered evidence that phosphorylation by GSK3ß is involved in the regulation of this process. In addition, while no function has been assigned to Trio in the cell cycle until now, we have found that Trio also interacts with proteins involved in centrosome function and cell cycle regulation. Following this finding, we have shown that Trio plays an unexpected role in late mitosis/cytokinesis. Altogether, these data open new avenues towards the understanding of Trio functions, by unveiling a pathway directly linking the RhoGEF Trio to the MT network. These data form the grounds of our project. The two partners, with complementary expertise (Partner 1, Debant’s lab, expertise in Rho GTPase signalling; Partner 2, Galjart’s lab, expertise in MT dynamics and +TIPs) have gathered solid data that support the feasibility of the project. We propose here to establish the interplay between the MT-binding ability of Trio and Trio-induced Rho GTPase signalling in the control of both neuronal differentiation and in cell cycle progression, using complementary approaches such as biochemistry, proteomics, cellular biology and live and high-resolution microscopy. Aim 1: We will focus on establishing the regulation of the MT-binding ability of Trio in the control of Rho GTPase signalling, by delineating the respective contribution of the different +TIP partners of Trio in the activation of its target GTPases in the context of neuronal differentiation. We will also test whether the MT-binding property of Trio affects MT-dynamics. Finally, we will characterise the upstream regulatory pathways that control the MT plus-end tracking property of Trio and investigate the molecular mechanisms underlying the role of GSK3ß on Trio function. Aim 2: We will decipher the role of Trio in cell cycle regulation. For this purpose, we will determine the molecular mechanisms by which Trio controls cytokinesis and whether Trio can coordinate MT dynamics and actin cytoskeleton remodelling in cell cycle regulation. We will also test whether Trio is regulated by mitotic kinases. Finally, we will test whether Trio’s effect on cytokinesis is conserved in other cellular contexts.